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Background: This study aimed to evaluate the dosimetric and radiobiological differences between 6MV flattened filter (FF) and flattening filter free (FFF) using volumetric modulated arc (VMAT) technique for head and neck (H&N) cancer patients. Materials and methods: Fifteen patients with H&N carcinoma were selected and treated with VMAT with FF (VMATFF) treatment plan. Retrospectively, additional VMAT treatment plans were developed using FFF beams (VMATFFF). Radiobiological parameters, such as equivalent uniform dose (EUD), tumor cure probability (TCP), and normal tissue complication probability (NTCP), were calculated using Niemierko's model for both VMATFF and VMATFFF. Correlation between dosimetric and radiobiological data were analyzed and compared. Results: The conformity index (CI) was 0.975 ± 0.014 (VMATFF) and 0.964 ± 0. 019 (VMATFFF) with p ≥ 0.05. Statistically, there was an insignificant difference in the planning target volume (PTV) results for TCP (%) values, with values of 81.20 ± 0.88% (VMATFF) and 81.01 ± 0.92 (%) (VMATFF). Similarly, there was an insignificant difference in the EUD (Gy) values, which were 71.53 ± 0.33 Gy (VMATFF) and 71.46 ± 0.34 Gy (VMATFFF). The NTCP values for the spinal cord, left parotid, and right parotid were 6.54 × 10-07%, 8.04%, and 7.69%, respectively, in the case of VMATFF. For VMATFFF, the corresponding NTCP values for the spinal cord, parotids left, and parotid right were 3.09 × 10-07%, 6.57%, and 6.73%, respectively. Conclusion: The EUD and Mean Dose to PTV were strongly correlated for VMATFFF. An increased mean dose to the PTV and greater TCP were reported for the VMATFF, which can enhance the delivery of the therapeutic dose to the target.
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Circulating DNAs are considered as degraded DNA fragments of approximately 50-200 bp, found in blood plasma, consisting of cell-free mitochondrial and nuclear DNA. Such cell-free DNAs in the blood are found to be altered in different pathological conditions including lupus, heart disease, and malignancies. While nuclear DNAs are being used and being developed as a powerful clinical biomarker in liquid biopsies, mitochondrial DNAs (mtDNAs) are associated with inflammatory conditions including cancer progression. Patients with cancer including prostate cancer are found to have measurable concentrations of mitochondrial DNA in circulation in comparison with healthy controls. The plasma content of mitochondrial DNA is dramatically elevated in both prostate cancer patients and mouse models treated with the chemotherapeutic drug. Cell-free mtDNA, in its oxidized form, induced a pro-inflammatory condition and activates NLRP3-mediated inflammasome formation which causes IL-1ß-mediated activation of growth factors. On the other hand, interacting with TLR9, mtDNAs trigger NF-κB-mediated complement C3a positive feedback paracrine loop and activate pro-proliferating signaling through upregulating AKT, ERK, and Bcl2 in the prostate tumor microenvironment. In this review, we discuss the growing evidence supporting cell-free mitochondrial DNA copy number, size, and mutations in mtDNA genes as potential prognostic biomarkers in different cancers and targetable prostate cancer therapeutic candidates impacting stromal-epithelial interactions essential for chemotherapy response.
Assuntos
Ácidos Nucleicos Livres , Neoplasias da Próstata , Humanos , Masculino , Animais , Camundongos , DNA Mitocondrial/metabolismo , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Mitocôndrias/metabolismo , Microambiente TumoralRESUMO
Telomere length is primarily controlled by the enzyme telomerase, but being chromatin structures, telomeres undergo epigenetic regulation for their maintenance and function. Altered telomere length among cancer cells combined with shorter telomere length in cancer-associated stromal cells, strongly implicated with progression to prostate cancer metastasis and cancer death and providing a novel target for therapeutics. Transforming growth factor-ß (TGF-ß) signaling pathways are well-recognized for their role in stromal-epithelial interactions responsible for prostate androgen responsiveness, promoting tumorigenesis. However, the underlying mechanism remains unclear. We sought to establish a role for TGF-ß in the regulation of telomere length in mouse and human prostate fibroblast. Polymerase chain reaction (PCR)-based telomere length measuring methods are widely used due to their repeatability and reproducibility. Using real-time RT-PCR-based telomere length measuring method, we identified that TGF-beta regulates telomere length via increased expression of histone methyltransferase, Suv39h1, which in turn affected histone methylation levels at the telomeric ends. Moreover, treatment of DAPT and non-steroidal antiandrogen bicalutamide demonstrated that notch and androgen signaling co-operated with TGF-ß in regulating stromal telomere length. Telomere variation in tumor cells and non-tumor cells within the tumor microenvironment greatly facilitates the clinical assessment of prostate cancer; therefore, understanding stromal telomere length regulation mechanism will hold significant prospects for cancer treatment, diagnosis, and prognosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03346-5.
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Chronic exposure to high glucose inside the human body helps in the progression of cancer by activating various signaling pathways including PI3K, Akt, mTOR, Ras, Raf, MAPK, and PKC. Hyperglycemia induces ROS and AGE production and decreases the functional activities of the cellular antioxidant system. By downregulating the prolyl hydroxylase, it stabilizes HIF-α leading to EMT-induced cancer progression and inhibition of apoptosis. High glucose level increases inflammation by creating a pro-inflammatory environment through the production of certain pro-inflammatory mediators (cytokines, chemokines, leukotrienes), and by influencing the recruitment of immune cells, leukocytes in the inflamed region. High glucose impairs the immune response and dysregulates ROS formation through the alteration in ETC and glutaminolysis which makes hyperglycemic patients more susceptible to viral infection. 2-DG is a modified form of D-glucose, that shows anticancer, anti-inflammatory, and anti-viral effects. It enters the cells through GLUT transporters and is converted into 2-deoxy-D-glucose-6-phosphate with the help of hexokinase. It inhibits the glycolysis, the TCA cycle, and the pentose phosphate pathway leading to ATP depletion. By downregulating glucose uptake and energy (ATP) production it halts various pathways responsible for cancer progression. It promotes the formation of anti-inflammatory mediators, and macrophage polarization, and also modulates immune function, which decreases inflammation. 2-DG inhibits PI3K/Akt/mTOR and upregulates the AMPK pathway, causing activation of the SIRT-4 gene that reduces lipogenesis, glucose uptake, nucleotide formation, and alters viral replication thus reducing the chances of infection.
Assuntos
Neoplasias , Viroses , Humanos , Glucose/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proliferação de Células , Glicólise , Neoplasias/tratamento farmacológico , Desoxiglucose/farmacologia , Inflamação , Trifosfato de Adenosina/metabolismoRESUMO
Head and neck cancers are often diagnosed at later stages with poor outcomes. Mesenchymal stem cells (MSC) are recruited to primary tumor sites where they can have pro- and antitumorigenic influence. In trying to better understand the dynamics between MSC and cancer cells, we found that head and neck cancer-MSC exposure resulted in mesenchymal features, elevated proliferation rate, and were more motile, like the same cells that fused with MSC. We orthotopically grafted the parental head and neck cancer cells, those fused with MSC, or those exposed to MSC into the tongues of mice. The cancer cells originally incubated with MSC developed larger more aggressive tumors compared to the parental cell line. RNA sequencing analysis revealed the expression of genes associated with drug resistance in the cancer cells exposed to MSC compared to parental cancer cells. Strikingly, MSC exposed cancer cell lines developed paclitaxel resistance that could be maintained up to 30 d after the initial co-incubation period. The secretory profile of the MSC suggested IL-6 to be a potential mediator of epigenetic imprinting on the head and neck cancer cells. When the MSC-imprinted cancer cells were exposed to the demethylation agent, 5-aza-2'deoxycytidine, it restored the expression of the drug resistance genes to that of parental cells. This study demonstrated that the recognized recruitment of MSC to tumors could impart multiple protumorigenic properties including chemotherapy resistance like that observed in the relatively rare event of cancer/MSC cell fusion.
Assuntos
Comunicação Celular , Resistencia a Medicamentos Antineoplásicos , Células-Tronco Mesenquimais/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica , Humanos , Camundongos , Modelos Biológicos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/etiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Stromal-epithelial interactions dictate cancer progression and therapeutic response. Prostate cancer (PCa) cells were identified to secrete greater concentration of mitochondrial DNA (mtDNA) compared to noncancer epithelia. Based on the recognized coevolution of cancer-associated fibroblasts (CAF) with tumor progression, we tested the role of cancer-derived mtDNA in a mechanism of paracrine signaling. We found that prostatic CAF expressed DEC205, which was not expressed by normal tissue-associated fibroblasts. DEC205 is a transmembrane protein that bound mtDNA and contributed to pattern recognition by Toll-like receptor 9 (TLR9). Complement C3 was the dominant gene targeted by TLR9-induced NF-κB signaling in CAF. The subsequent maturation complement C3 maturation to anaphylatoxin C3a was dependent on PCa epithelial inhibition of catalase in CAF. In a syngeneic tissue recombination model of PCa and associated fibroblast, the antagonism of the C3a receptor and the fibroblastic knockout of TLR9 similarly resulted in immune suppression with a significant reduction in tumor progression, compared to saline-treated tumors associated with wild-type prostatic fibroblasts. Interestingly, docetaxel, a common therapy for advanced PCa, further promoted mtDNA secretion in cultured epithelia, mice, and PCa patients. The antiapoptotic signaling downstream of anaphylatoxin C3a signaling in tumor cells contributed to docetaxel resistance. The inhibition of C3a receptor sensitized PCa epithelia to docetaxel in a synergistic manner. Tumor models of human PCa epithelia with CAF expanded similarly in mice in the presence or absence of docetaxel. The combination therapy of docetaxel and C3 receptor antagonist disrupted the mtDNA/C3a paracrine loop and restored docetaxel sensitivity.
Assuntos
Anafilatoxinas/metabolismo , Fibroblastos Associados a Câncer/patologia , DNA Mitocondrial/metabolismo , Docetaxel/farmacologia , Resistencia a Medicamentos Antineoplásicos , Epitélio/patologia , Neoplasias da Próstata/patologia , Animais , Antineoplásicos/farmacologia , Apoptose , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Comunicação Parácrina , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Receptor Toll-Like 9/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Periodontal diseases can lead to chronic inflammation affecting the integrity of the tooth supporting tissues. Recently, a striking association has been made between periodontal diseases and primary cancers in the absence of a mechanistic understanding. Here we address the effect of periodontal inflammation (PI) on tumor progression, metastasis, and possible underlining mechanisms. We show that an experimental model of PI in mice can promote lymph node (LN) micrometastasis, as well as head and neck metastasis of 4T1 breast cancer cells, both in early and late stages of cancer progression. The cervical LNs had a greater tumor burden and infiltration of MDSC and M2 macrophages compared with LNs at other sites. Pyroptosis and the resultant IL-1ß production were detected in patients with PI, mirrored in mouse models. Anakinra, IL-1 receptor antagonist, limited metastasis, and MDSC recruitment at early stages of tumor progression, but failed to reverse established metastatic tumors. PI and the resulting production of IL-1ß was found to promote CCL5, CXCL12, CCL2, and CXCL5 expression. These chemokines recruit MDSC and macrophages, finally enabling the generation of a premetastatic niche in the inflammatory site. These findings support the idea that periodontal inflammation promotes metastasis of breast cancer by recruiting MDSC in part by pyroptosis-induced IL-1ß generation and downstream CCL2, CCL5, and CXCL5 signaling in the early steps of metastasis. These studies define the role for IL-1ß in the metastatic progression of breast cancer and highlight the need to control PI, a pervasive inflammatory condition in older patients.
Assuntos
Neoplasias da Mama/complicações , Neoplasias da Mama/patologia , Células Mieloides/patologia , Doenças Periodontais/complicações , Animais , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Interleucina-1beta/metabolismo , Camundongos , Metástase Neoplásica , PiroptoseRESUMO
Genomic changes that drive cancer initiation and progression contribute to the co-evolution of the adjacent stroma. The nature of the stromal reprogramming involves differential DNA methylation patterns and levels that change in response to the tumor and systemic therapeutic intervention. Epigenetic reprogramming in carcinoma-associated fibroblasts are robust biomarkers for cancer progression and have a transcriptional impact that support cancer epithelial progression in a paracrine manner. For prostate cancer, promoter hypermethylation and silencing of the RasGAP, RASAL3 that resulted in the activation of Ras signaling in carcinoma-associated fibroblasts. Stromal Ras activity initiated a process of macropinocytosis that provided prostate cancer epithelia with abundant glutamine for metabolic conversion to fuel its proliferation and a signal to transdifferentiate into a neuroendocrine phenotype. This epigenetic oncogenic metabolic/signaling axis seemed to be further potentiated by androgen receptor signaling antagonists and contributed to therapeutic resistance. Intervention of stromal signaling may complement conventional therapies targeting the cancer cell.
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Fibroblastos Associados a Câncer , Epigênese Genética , Neoplasias/genética , Neoplasias/metabolismo , Animais , Diferenciação Celular , Cromatina/metabolismo , Metilação de DNA , Histonas/metabolismo , Humanos , PinocitoseRESUMO
Heterogeneous prostatic carcinoma-associated fibroblasts (CAF) contribute to tumor progression and resistance to androgen signaling deprivation therapy (ADT). CAF subjected to extended passaging, compared to low passage CAF, were found to lose tumor expansion potential and heterogeneity. Cell surface endoglin (CD105), known to be expressed on proliferative endothelia and mesenchymal stem cells, was diminished in high passage CAF. RNA-sequencing revealed SFRP1 to be distinctly expressed by tumor-inductive CAF, which was further demonstrated to occur in a CD105-dependent manner. Moreover, ADT resulted in further expansion of the CD105+ fibroblastic population and downstream SFRP1 in 3-dimensional cultures and patient-derived xenograft tissues. In patients, CD105+ fibroblasts were found to circumscribe epithelia with neuroendocrine differentiation. CAF-derived SFRP1, driven by CD105 signaling, was necessary and sufficient to induce prostate cancer neuroendocrine differentiation in a paracrine manner. A partially humanized CD105 neutralizing antibody, TRC105, inhibited fibroblastic SFRP1 expression and epithelial neuroendocrine differentiation. In a novel synthetic lethality paradigm, we found that simultaneously targeting the epithelia and its microenvironment with ADT and TRC105, respectively, reduced castrate-resistant tumor progression, in a model where either ADT or TRC105 alone had little effect.
Assuntos
Diferenciação Celular , Endoglina/metabolismo , Fibroblastos/metabolismo , Proteínas de Neoplasias/metabolismo , Células Neuroendócrinas/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Endoglina/genética , Fibroblastos/patologia , Humanos , Masculino , Proteínas de Neoplasias/genética , Células Neuroendócrinas/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologiaRESUMO
We investigate the physicochemical interactions of gold nanorod (GNR) with single-stranded, double-stranded, and hairpin DNA structures to improve the biological compatibility as well as the therapeutic potential, including the photothermal effect of the conjugates. Studies have demonstrated that different DNA secondary structures, containing thiol group, have different patterns of physicochemical interaction. Conjugation efficiency of paired oligonucleotides are significantly higher than that of oligonucleotides with naked bases. Furthermore, hairpin-shaped DNA structures are most efficient in terms of conjugation and increased dispersion, with least interference on GNR near-infrared absorbance and photothermal effect. Our conjugation method can successfully exchange the overall coating of the GNR, attaching the maximum number of DNA molecules, thus far reported. Chemical mapping depicted uniform attachment of thiolated DNA molecules without any topological preference on the GNR surface. Hairpin DNA-coated GNR are suitable for intracellular uptake and remain dispersed in the cellular environment. Finally, we conjugated GNR with 5-fluoro-2'-deoxyuridine-containing DNA hairpin and the conjugate demonstrated significant cytotoxic activity against human cervical cancer cell line (KB). Thus, hairpin DNA structures could be utilized for optimal dispersion and photothermal effect of GNR, along with the delivery of cytotoxic nucleotides, developing the concept of multimodality approach.
RESUMO
Prostate cancer is an androgen-dependent disease subject to interactions between the tumor epithelium and its microenvironment. Here, we found that epigenetic changes in prostatic cancer-associated fibroblasts (CAF) initiated a cascade of stromal-epithelial interactions. This facilitated lethal prostate cancer growth and development of resistance to androgen signaling deprivation therapy (ADT). We identified a Ras inhibitor, RASAL3, as epigenetically silenced in human prostatic CAF, leading to oncogenic Ras activity driving macropinocytosis-mediated glutamine synthesis. Interestingly, ADT further promoted RASAL3 epigenetic silencing and glutamine secretion by prostatic fibroblasts. In an orthotopic xenograft model, subsequent inhibition of macropinocytosis and glutamine transport resulted in antitumor effects. Stromal glutamine served as a source of energy through anaplerosis and as a mediator of neuroendocrine differentiation for prostate adenocarcinoma. Antagonizing the uptake of glutamine restored sensitivity to ADT in a castration-resistant xenograft model. In validating these findings, we found that prostate cancer patients on ADT with therapeutic resistance had elevated blood glutamine levels compared with those with therapeutically responsive disease (odds ratio = 7.451, P = 0.02). Identification of epigenetic regulation of Ras activity in prostatic CAF revealed RASAL3 as a sensor for metabolic and neuroendocrine reprogramming in prostate cancer patients failing ADT.
Assuntos
Inativação Gênica , Glutamina/metabolismo , Proteínas de Neoplasias/metabolismo , Tumores Neuroendócrinos/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Fibroblastos/metabolismo , Fibroblastos/patologia , Glutamina/genética , Humanos , Masculino , Camundongos , Proteínas de Neoplasias/genética , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Radiation therapy is the primary intervention for nearly half of the patients with localized advanced prostate cancer and standard of care for recurrent disease following surgery. The development of radiation-resistant disease is an obstacle for nearly 30-50% of patients undergoing radiotherapy. A better understanding of mechanisms that lead to radiation resistance could aid in the development of sensitizing agents to improve outcome. Here we identified a radiation-resistance pathway mediated by CD105, downstream of BMP and TGF-ß signaling. Antagonizing CD105-dependent BMP signaling with a partially humanized monoclonal antibody, TRC105, resulted in a significant reduction in clonogenicity when combined with irradiation. In trying to better understand the mechanism for the radio-sensitization, we found that radiation-induced CD105/BMP signaling was sufficient and necessary for the upregulation of sirtuin 1 (SIRT1) in contributing to p53 stabilization and PGC-1α activation. Combining TRC105 with irradiation delayed DNA damage repair compared to irradiation alone. However, in the absence of p53 function, combining TRC105 and radiation resulted in no reduction in clonogenicity compared to radiation alone, despite similar reduction of DNA damage repair observed in p53-intact cells. This suggested DNA damage repair was not the sole determinant of CD105 radio-resistance. As cancer cells undergo an energy deficit following irradiation, due to the demands of DNA and organelle repair, we examined SIRT1's role on p53 and PGC-1α with respect to glycolysis and mitochondrial biogenesis, respectively. Consequently, blocking the CD105-SIRT1 axis was found to deplete the ATP stores of irradiated cells and cause G2 cell cycle arrest. Xenograft models supported these findings that combining TRC105 with irradiation significantly reduces tumor size over irradiation alone (p value = 10-9). We identified a novel synthetic lethality strategy of combining radiation and CD105 targeting to address the DNA repair and metabolic addiction induced by irradiation in p53-functional prostate cancers.
Assuntos
Endoglina/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/radioterapia , Tolerância a Radiação/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Nus , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/efeitos da radiação , Neoplasias da Próstata/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Hemorrhagic cystitis is an inflammatory and ulcerative bladder condition associated with systemic chemotherapeutics, like cyclophosphomide. Earlier, we reported reactive oxygen species resulting from cyclophosphamide metabolite, acrolein, causes global methylation followed by silencing of DNA damage repair genes. Ogg1 (8-oxoguanine DNA glycosylase) is one such silenced base excision repair enzyme that can restore DNA integrity. The accumulation of DNA damage results in subsequent inflammation associated with pyroptotic death of bladder smooth muscle cells. We hypothesized that reversing inflammasome-induced imprinting in the bladder smooth muscle could prevent the inflammatory phenotype. Elevated recruitment of Dnmt1 and Dnmt3b to the Ogg1 promoter in acrolein treated bladder muscle cells was validated by the pattern of CpG methylation revealed by bisulfite sequencing. Knockout of Ogg1 in detrusor cells resulted in accumulation of reactive oxygen mediated 8-Oxo-dG and spontaneous pyroptotic signaling. Histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), restored Ogg1 expression in cells treated with acrolein and mice treated with cyclophosphamide superior to the standard of care, mesna or nicotinamide-induced DNA demethylation. SAHA restored cyclophosphamide-induced bladder pathology to that of untreated control mice. The observed epigenetic imprinting induced by inflammation suggests a new therapeutic target for the treatment of hemorrhagic cystitis.
Assuntos
Ciclofosfamida/toxicidade , Cistite/etiologia , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Acroleína/toxicidade , Animais , Células Cultivadas , Cistite/induzido quimicamente , Cistite/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/deficiência , DNA Glicosilases/genética , Regulação para Baixo/efeitos dos fármacos , Feminino , Mesna/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Niacinamida/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Vorinostat , DNA Metiltransferase 3BRESUMO
Muscle inflammation is often associated with its expansion. Bladder smooth muscle inflammation-induced cell death is accompanied by hyperplasia and hypertrophy as the primary cause for poor bladder function. In mice, DNA damage initiated by chemotherapeutic drug cyclophosphamide activated caspase 1 through the formation of the NLRP3 complex resulting in detrusor hyperplasia. A cyclophosphamide metabolite, acrolein, caused global DNA methylation and accumulation of DNA damage in a mouse model of bladder inflammation and in cultured bladder muscle cells. In correlation, global DNA methylation and NLRP3 expression was up-regulated in human chronic bladder inflammatory tissues. The epigenetic silencing of DNA damage repair gene, Ogg1, could be reversed by the use of demethylating agents. In mice, demethylating agents reversed cyclophosphamide-induced bladder inflammation and detrusor expansion. The transgenic knock-out of Ogg1 in as few as 10% of the detrusor cells tripled the proliferation of the remaining wild type counterparts in an in vitro co-culture titration experiment. Antagonizing IL-1ß with Anakinra, a rheumatoid arthritis therapeutic, prevented detrusor proliferation in conditioned media experiments as well as in a mouse model of bladder inflammation. Radiation treatment validated the role of DNA damage in the NLRP3-associated caspase 1-mediated IL-1ß secretory phenotype. A protein array analysis identified IGF1 to be downstream of IL-1ß signaling. IL-1ß-induced detrusor proliferation and hypertrophy could be reversed with the use of Anakinra as well as an IGF1 neutralizing antibody. IL-1ß antagonists in current clinical practice can exploit the revealed mechanism for DNA damage-mediated muscular expansion.
Assuntos
Hiperplasia/metabolismo , Inflamação/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Interleucina-1beta/metabolismo , Músculo Liso/patologia , Animais , Apoptose/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/metabolismo , Humanos , Hiperplasia/patologia , Inflamação/genética , Inflamação/patologia , Fator de Crescimento Insulin-Like I/genética , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Camundongos , Músculo Liso/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transdução de Sinais/efeitos dos fármacos , Bexiga Urinária/metabolismo , Bexiga Urinária/patologiaRESUMO
The vast majority of cases of infectious cystitis are easily treated, and most patients have no long-term complications. However, hemorrhagic cystitis is a potentially deadly complication associated with pelvic radiation therapy, chemotherapy, and stem-cell transplant therapy. The focus of current understanding, and hence therapy, is directed toward urothelial cell death. However, the primary functional ramification of inflammatory bladder disease is the loss of compliance due to muscular expansion. Recent studies on smooth muscle response in models of bladder inflammation demonstrate a process of pyroptotic cell death that potentiates further muscle hyperplasia. These findings may support alternative interventions for subjects with hemorrhagic cystitis refractive to current therapy.
RESUMO
The budding yeast transcriptional repressor Sum1p binds to several promoters and recruits Hst1p, an NAD(+)-dependent histone deacetylase, at these promoters with the help of another protein Rfm1p. Hst1p causes repression of transcription by histone deacetylation of chromatin at its target promoters. In an earlier work we have shown that about 13-fold increase in Sum1p levels, brought about by expressing SUM1 from the high copy 2 micron plasmid (2 µ-SUM1), suppressed cold-sensitive growth phenotype associated with mutations in the α-tubulin gene TUB1. In this work we show that the dosage suppression is accompanied by an elevation of α-tubulin levels in mutant cells at their non-permissive growth temperature of 14°C. Further, 2 µ-SUM1 significantly rescued the benomyl-supersensitive growth phenotype of mutant cells having wild-type tubulin subunits but a deficiency in tubulin folding cofactors. Finally, wild-type 2 µ-SUM1 transformants, having no known mutation in microtubule-related genes, displayed spindle microtubules which were substantially more stable than of wild-type control cells when challenged with microtubule-depolymerizing drugs. Therefore, we conclude that high copies of Sum1p stabilize microtubules against a variety of adverse and destabilizing conditions like mutations, low temperatures and drugs.
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Dosagem de Genes , Microtúbulos/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico , Microtúbulos/ultraestrutura , Mutação , Dobramento de Proteína , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Supressão Genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
The budding yeast protein Sum1 is a transcription factor that associates with the histone deacetylase Hst1p or, in its absence, with Sir2p to form repressed chromatin. In this study, SUM1 has been identified as an allele-specific dosage suppressor of mutations in the major alpha-tubulin-coding gene TUB1. When cloned in a 2mu vector, SUM1 suppressed the cold-sensitive and benomyl-hypersensitive phenotypes associated with the tub1-1 mutation. The suppression was Hst1p- and Sir2p-independent, suggesting that it was not mediated by deacetylation events associated with Sum1p when it functions along with its known partner histone deacetylases. This protein was confined to the nucleus, but did not colocalize with the microtubules nor did it bind to alpha- or beta-tubulin. Cells deleted of SUM1 showed hypersensitivity to benomyl and cold-sensitive growth, phenotypes exhibited by mutants defective in microtubule function and cytoskeletal defects. These observations suggest that Sum1p is a novel regulator of microtubule function. We propose that as a dosage suppressor, Sum1p promotes the formation of microtubules by increasing the availability of the alphabeta-heterodimer containing the mutant alpha-tubulin subunit.
Assuntos
Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Benomilo/toxicidade , Temperatura Baixa , Deleção de Genes , Dosagem de Genes , Expressão Gênica , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
This study reports the presence of a high molecular weight protein (Bengalin) from the Indian black scorpion (Heterometrus bengalensis) venom having antiosteoporosis activity in experimental osteoporosis developed in female albino Wister rats. Bengalin was purified through DEAE-cellulose ion exchange chromatography and high performance liquid chromatography. The molecular weight of the Bengalin was found to be 72kDa and the first 20 amino acid sequence was found to be G-P-L-T-I-L-H-I-N-D-V-H-A-A/R-F-E-Q/G-F/G-N-T. Bengalin exhibited significant antiosteoporosis activity in experimental female rats, which was confirmed through analysis of urine Ca(2+), PO(4)(3-), CRE & OH-P. Bengalin (3 microg and 5 microg/100g rat/i.p.) antagonized osteoporosis by restoring urinary Ca(2+), PO(4)(3-), CRE and OH-P, serum/plasma Ca(2+), PO(4)(3-), ALP, TRAP, PTH, T(3), TSH, Osteocalcin, IL1, IL6 and TNF alpha and bone minerals Ca(2+), P, Mg(2+), Zn(2+), Na(+), as compared with the sham operated control rats. Bone minerals density of osteoporosis female rats was improved due to Bengalin, observed through DEXA scan. Subacute toxicity studies in male albino mice, Bengalin showed cardiotoxicity. In vivo experiments, Bengalin showed cardiotoxicity on isolated guinea pig heart, guinea pig auricle, and neurotoxicity on isolated rat phrenic nerve diaphragm preparation. Further detail studies on the toxicity, antiosteoporosis and structural identity of Bengalin are warranted.
Assuntos
Conservadores da Densidade Óssea , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Escorpiões/fisiologia , Sequência de Aminoácidos , Animais , Densidade Óssea/efeitos dos fármacos , Cromatografia DEAE-Celulose , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Feminino , Cobaias , Coração/efeitos dos fármacos , Índia , Masculino , Camundongos , Dados de Sequência Molecular , Peso Molecular , Osteoporose/patologia , Osteoporose/prevenção & controle , Nervo Frênico/efeitos dos fármacos , Proteínas/química , Ratos , Ratos Wistar , Venenos de Escorpião/toxicidadeRESUMO
The present study was designed to explore the antiosteoporosis activity of the Indian black scorpion (Heterometrus bengalensis) venom on experimental osteoporosis female albino rats. Sham operated control rats were designated as Gr I, Gr II animals served as osteoporosis control, Gr III osteoporosis rats were treated with SV (1/25th of MLD), Gr IV osteoporosis rats were treated with 1/50th of MLD of SV and Gr V osteoporosis rats were treated with standard (calcium and vit-D3). As compared with the Gr I rats, the Gr II rats showed typical osteoporosis changes in increased of urinary Ca(2+), PO(4)(3-), CRE, OH-P levels, serum/plasma Ca(2+), PO(4)(3-), TRAP, IL1, IL6, TNFalpha and PTH level, bone Ca(2+), PO(4)(3-), Mg(2+), Zn(2+) and decreased level of serum/plasma ALP, EST and PTH, bone Na(+). In Gr III, Gr IV and Gr V rats, the osteoporosis changes of urine, serum and bone, were significantly restored as compared with the Gr II rats. The bone dimensions, morphology and histological changes observed in Gr II rats were restored in Gr III, Gr IV and Gr V rats. This study confirms that the Indian black scorpion venom may influence bone remodeling process by stimulating bone formation and reducing bone resorption process of osteogenesis.